Actinomycetes are important microorganisms which have found wide-spread utility in the microbiological industry, as microorganisms for producing the useful substances such as antibiotics.
It is extremely beneficial from the industrial standpoint to use the recombinant DNA technology for the breeding of actinomycetes or for the elucidation of the genetic characteristics of actinomycetes.
From such a viewpoint, many plasmid vectors which are usable for actinomycetes have been constructed and reported. It has however been found that when a gene of actinomycetes is inserted in these known plasmid vectors, the gene thus inserted is not always expressed as desired and the expression of the gene is not affected by any artificial control. The conventional actinomycetes plasmid vectors were, therefore, not fully satisfactory for such a genetic engineering procedure which requires a skill of high order for the control of the expression of genes.
The present inventors have made investigations in an attempt to make improvements in these problems. As a result, we have now succeeded in isolating such a novel DNA fragment which contains the streptomycin resistance
microorganism, Streptomyces griseus ISP 5236 (ATCC gene of a 23345), a known streptomycin-producing strain, and which also contains a DNA region possessing a function to control the expression of said gene. It has also been found that even when the streptomycin resistance gene is introduced into a plasmid vector with omitting the DNA region which possesses the function to control the expression of the streptomycin resistance gene, in the resulting plasmid vector recombinant DNA molecule, the streptomycin resistance gene introduced is not expressed as desired.
The present inventors have already attempted the isolation of the streptomycin resistance gene from the above-mentioned Streptomyces griseus ISP 5236 known as the streptomycin-producing microorganism, as well as the cloning of said gene. As a result of the experiments, the location and characteristics of the streptomycin resistance gene were elucidated to some extent (Japanese Patent Application No. 265373/79 filed Dec. 18, 1979; see Japanese Patent Application first publication "Kokai" No. 146186/86).
Thus, the streptomycin resistance gene has already been found by the present inventors to have the following characteristics:
(a) The gene is comprised in a Bgl II-Bgl II DNA fragment of a length of about 7.0 kilobose (kb) which is obtained by cutting or excising the DNA of the chromosome which governs the streptomycin resistance of Streptomyces griseus, with a restriction endonuclease Bgl II.
(b) The restriction endonuclease map of the above DNA fragment of a length of about 7.0 kb, which contains said gene, is as shown in FIG. 1 of the accompanying drawings.
(c) The gene encodes a phosphotransferase, the enzyme which phosphorylates the 6-OH group of an aminoglycoside antibiotic, notably, streptomycin.
The present inventors have also found that the above streptomycin resistance gene locates in such a DNA fragment which is obtained by cleaving with a restriction endonuclease Sph I, the above-mentioned Bgl II-Bgl II DNA fragment of about 7.0 kb in length, which had, in turn, been obtained by excising the chromosome DNA of Streptomyces griseus with the restriction endonuclease Bgl II (see Japanese Patent Application first publication "Kokai" No. 146186/86 referred to above).
Further, the present inventors also have succeeded in constructing a novel hybrid plasmid by insertion of said Bgl II-Bgl II DNA fragment of a length of about 7.0 kb which contains the streptomycin resistance gene ("streptomycin" is hereinafter abbreviated as "SM"), into the restriction endonuclease Bgl II-cleavage site of the DNA of a known plasmid pIJ 702, in accordance with a known gene recombination technique. This novel hybrid plasmid was named "pST 141". It was also found that the plasmid pST 141 has a length of about 12.6 kb, and the restriction endonuclease map of this hybrid plasmid pST 141 is as illustrated in FIG. 2 of the accompanying drawings (see the above-mentioned Japanese Patent Application first publication "Kokai"). A Streptomyces lividans transformant containing the plasmid pST 141 has been deposited under FERM P-7984 since Dec. 6, 1984 with "Fermentation Research Institute, Agency of Industrial Science of Technology" located at No. 1-3 Higashi 1-chome, Yatabe-machi Tsukuba-shi, Ibarakiken, Japen (305), and it has also been deposited there under FERM BP-1198 through the procedure of transfer which was done in accordance with the provisions of the International Deposit prescribed in the Budapest Treaty.
An invention was thus completed and claimed in Japanese Patent Application No. 265373/79, which relates to "A DNA segment containing a streptomycin resistance gene therein, characterized in that said segment is equivalent to such a DNA fragment which has been originated from the DNA of Streptomyces griseus and which is located in a hybrid plasmid pST 141; said hybrid plasmid pST 141 being a hybrid plasmid composed of a Bgl II fragment of the DNA of Streptomyces griseus and of a Bgl II fragment of the DNA of a plasmid pIJ 702; and said plasmid pST 141 having the restriction endonuclease cleavage sites shown in FIG. 2 of the accompanying drawings and also having a length of about 12.6 kb".
The present inventors have now proceeded with a further investigation. Thus, the above-described Bgl II-Bgl II DNA fragment which has been obtained by excising the DNA of the chromosome of Streptomyces griseus with the enzyme Bgl II, has the restriction endonuclease map of FIG. 1, contains the SM resistance gene and has a length of about 7.0 kb is now further excised with a restriction endonuclease Sph I to give a Sph I-Sph I fragment. The resulting Sph I-Sph I fragment is then excised with a restriction endonuclease Pst I to give a Sph I-Pst I fragment, or the resulting Sph I-Sph I fragment is then excised with a restriction endonuclease Bam HI to give a Sph I-Bam HI fragment. The Sph I-Pst I fragment and the Sph I-Bam HI fragment so obtained are used separately and inserted into various vectors so that various types of plasmids are reconstructed. The various types of plasmids thus obtained are then introduced separately into cells of such a strain of Streptomyces lividans which intrinsically does not produce streptomycin. Various kinds of the resultant transformants of Streptomyces lividans are separately cultured. Streptomycin is added to the culture broth of the respective transformants, followed by further incubation. From the states of growth of the individual transformants in the incubation experiments, it is examined whether or not a streptomycin-inactivating enzyme is produced by the individual transformants. Further, it is also investigated from the results of the above examination whether or not the SM resistance gene present in the plasmids as introduced is expressed. As a result of the above investigations, it has been found that such strains which do not or little show the expression of the SM resistance gene are occurring, in addition to the strains which show the SM resistance, even when the so incubated strains are such those that have been transformed just by the plasmids containing the entire DNA sequence which is corresponding to the SM resistance gene. With the foregoing in view, a further investigation has been continued. As a result, it has now been found that the above-described Sph I DNA fragment of a length of about 3.8 kb, which is obtained by excising with the enzyme Sph I the aforementioned Bgl II-Bgl II fragment of a length of about 7.0 kb having the restriction endonuclease map of FIG. 1 and containing the SM resistance gene, does contain such DNA region which possesses the function to control the expression of the SM resistance gene, namely, such a DNA region which is capable of sensing the existence of streptomycin as added artificially from the outside and capable of governing, i.e., controlling the expression of the SM resistance gene and also that the SM resistance gene is not expressed as desired unless the DNA region possessing the function to control the expression of the gene is existing in the vicinity of the SM resistance gene. Some part of these finding have already been reported by the present inventors in "The Journal of Antibiotics", Vol. 39, No. 10, 1505-1507 (published Oct. 25, 1986).
The present inventors have hence expected that a recombinant plasmid capable of reliably acting as a selected marker of the SM resistance is possible to be constructed by inserting into a plasmid vector said SM resistance gene, in association with such DNA fragment consisting of the above-mentioned DNA region which possesses the function to control the expression of the SM resistance gene, or a DNA fragment comprising said DNA region, in case such DNA fragment can be isolated and recovered. Moreover, such a host strain as transformed by the recombinant plasmid which contains the SM resistance gene as well as the DNA fragment (region) possessing the function to control the expression of the SM resistance gene will be able to show the expression of the SM resistance gene as desired artificially when a substance capable of inducing the expression of the SM resistance gene, for example, streptomycin is added externally. It is also expected that when the above-mentioned DNA fragment (or region) possessing the function to control the SM resistance gene, as well as an appropriate gene in place of the SM resistance gene are inserted into a plasmid according to a DNA recombinant technique and when the recombinant plasmid so obtained is introduced into a host cell of actinomycetes, said appropriate gene is possible to be expressed as desired in the transformant of the host actinomycetes cell. Therefore, the DNA fragment having the function as above described is industrially useful in various aspects. The present inventors have now succeeded in obtaining such a novel DNA fragment.